Skip to main content

A high throughput targeted and non-targeted method for the analysis of microcystins and anatoxin-A using on-line solid phase extraction coupled to liquid chromatography–quadrupole time-of-flight high resolution mass spectrometry


Microcystins are cyclic heptapeptide hepatotoxins produced by cyanobacteria in freshwater. Sample preparation for the analysis of these cyanotoxins in water from algal blooms can take up to several days due to the matrix complexity and the low detection limits required to comply with current legislation. Moreover, there is a large number of unknown microcystins that could potentially exist in the environment resulting from different amino acid substitutions into the microcystin skeletal structure. To tackle these problems, the present study involved the development of a high throughput method based on on-line solid phase extraction coupled to liquid chromatography that could provide quantitative results for 12 microcystin variants (LR, YR, RR, HtyR, HilR, WR, LW, LA, LF, LY, Dha7-LR, and Dha7-RR) and anatoxin-A in less than 3 h with detection limits between 0.004 and 0.01 μg L−1 and expanded uncertainty between 4 and 14%. Data-dependent acquisition was employed for the non-targeted analysis of these cyanotoxins. Filtering the data based on structure diagnostic fragments, two unknown microcystin variants not previously reported in the literature were detected. The structures Leu1-microcystin-Met(O)R and Leu1-microcystin-LY were fully characterized by accurate mass measurement, collision-induced dissociation, and fragmentation prediction software.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Loftin KA, Graham JL, Hilborn ED, Lehmann SC, Meyer MT, Dietze JE, Griffith CB. Cyanotoxins in inland lakes of the United States: occurrence and potential recreational health risks in the EPA National Lakes Assessment 2007. Harmful Algae. 2016;56:77–90.

    Article  CAS  Google Scholar 

  2. Catherine A, Bernard C, Spoof L, Bruno M. Microcystins and nodularins. In: Meriluoto J, Spoof S, Codd GA, editors. Handbook of cyanobacterial monitoring and cyanotoxin analysis, vol. 11; 2017. p. 109–18.

    Google Scholar 

  3. Yuan M, Carmichael WW, Hilborn ED. Microcystin analysis in human sera and liver from human fatalities in Caruaru, Brazil 1996. Toxicon. 2008;48:627–40.

    Article  Google Scholar 

  4. Azevedo S, Carmichael WW, Jochimsen EM, Rinehart KL, Lau S, Shaw GR, Eaglesham GK. Human intoxication by microcystins during renal dialysis treatment in Caruaru—Brazil. Toxicology. 2002;181-182:441–6.

    Article  CAS  Google Scholar 

  5. Carmichael WW, Boyer GL. Health impacts from cyanobacteria harmful algae blooms: implications for the North American Great Lakes. Harmful Algae. 2016;54:194–212.

    Article  Google Scholar 

  6. Osswald J, Rellán S, Gago A, Vasconcelos V. Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, anatoxin-a. Environ Int. 2007;33:1070–89.

    Article  CAS  Google Scholar 

  7. Niedermeyer T. Microcystin congeners described in the literature. Available online: doi:10.6084/m9.figshare.880756. Accessed 13 Feb 2017.

  8. Cyanobacterial toxins: microcystin-LR in drinking-water. Background document for development of WHO guidelines for drinking-water quality. Originally published in Guidelines for drinking-water quality, 2nd ed. Addendum to Vol. 2. Health criteria and other supporting information. World Health Organization, Geneva; 1998.

  9. Ontario drinking water quality standards, schedule 2, chemical standards, Ontario Safe Drinking Water Act; 2002.

  10. Samdal IA, Ballot A, Lovberg KE, Miles CO. Multihapten approach leading to a sensitive ELISA with broad cross-reactivity to microcystins and nodularin. Environ. Sci. Technol. 2014;48(14):8035–43.

    Article  CAS  Google Scholar 

  11. Miles CO, Sandvik M, Hezron EN, Rundberget T, Alistair L, Rise F, Ballot A. Thiol derivatization for LC-MS identification of microcystins in complex matrices. Environ Sci Technol. 2012;46(12):8937–44.

    Article  CAS  Google Scholar 

  12. Shoemaker JA, Tettenhorst DR, de la Cruz A. EPA method 544: determination of microcystins and nodularin in drinking water by solid phase extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS). EPA Document #: EPA/600/R-14/474. 2015, version 1.0.

  13. Taguchi V, Jenkins S. The determination of microcystins and anatoxin-A in water by liquid chromatography–(electrospray ionization) tandem mass spectrometry [LC-(ESI) MS/MS]. Ministry of the Environment and Climate Change, LaSB Method E3450. 2016, version 5.1.

  14. Mashile GP, Nomngongo PN. Recent application of solid phase based techniques for extraction and preconcentration of cyanotoxins in environmental matrices. Cr rev Anal Chem. 2017;47(2):119–26.

    Article  CAS  Google Scholar 

  15. Foss AJ, Aubel MT. Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS). Toxicon. 2015;104:91–101.

    Article  CAS  Google Scholar 

  16. Kaloudir T, Zervou SK, Tsimeli K, Triantis TM, Fotiou T, Hiskia A. Determination of microcystins and nodularin (cyanobacterial toxins) in water by LC–MS/MS monitoring of Lake Marathonas, a water reservoir of Athens, Greece. J Hazard Mater. 2013;263:105–15.

    Article  Google Scholar 

  17. Balest L, Murgolo S, Sciancalepore L, Montemurro P, Abis PP, Pastore C, Mascolo G. Ultra-trace levels analysis of microcystins and nodularin in surface water by on-line solid-phase extraction with high-performance liquid chromatography tandem mass spectrometry. Anal Bioanal Chem. 2016;408:4063–71.

    Article  CAS  Google Scholar 

  18. Fayad PB, Roy-Lachapelle A, Duy SV, Prévost M, Sauvé S. On-line solid-phase extraction coupled to liquid chromatography tandem mass spectrometry for the analysis of cyanotoxins in algal blooms. Toxicon. 2015;108:167–75.

    Article  CAS  Google Scholar 

  19. Beltrán E, Ibáñez M, Sancho JV, Hernández F. Determination of six microcystins and nodularin in surface and drinking waters by on-line solid phase extraction–ultra high pressure liquid chromatography tandem mass spectrometry. J. Chrom. A. 2012;1266:61–8.

    Article  Google Scholar 

  20. Lee HS, Jeong CK, Lee HM, Choi SJ, Do KS, Kim K, Kim YH. On-line trace enrichment for the simultaneous determination of microcystins in aqueous samples using high-performance liquid chromatography with diode-array detection. J Chrom A. 1999; 179–84.

  21. Shan Y, Shi X, Dou A, Zou C, He H, Yang Q, Zhao S, Lu X, Xu G. A fully automated system with on-line micro solid-phase extraction combined with capillary liquid chromatography–tandem mass spectrometry for high throughput analysis of microcystins and nodularin-R in tap water and lake water. J. Chrom. A. 2011;1218:1743–8.

    Article  CAS  Google Scholar 

  22. Ortielli D, Edder P, Cognard E, Jan P. Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry. Anal Chim Acta. 2008; 230–37.

  23. Flores C, Caixach J. An integrated strategy for rapid and accurate determination of free and cell-bound microcystins and related peptides in natural blooms by liquid chromatography–electrospray-high resolution mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry using both positive and negative ionization modes. J Chrom a. 2015;1407:76–89.

    Article  CAS  Google Scholar 

  24. Hollingdale C, Thomas K, Lewis N, Békri K, McCarron P, Quilliam MA. Feasibility study on production of a matrix reference material for cyanobacterial toxins. Anal Bioanal Chem. 2015;407:5353–63.

    Article  CAS  Google Scholar 

  25. Kim IS, Nguyen GH, Kim S, Lee J, Yu HW. Evaluation of methods for cyanobacterial cell lysis and toxin (microcystin-LR) extraction using chromatographic and mass spectrometric analyses. Environ Eng res. 2009;14(4):250–4.

    Article  Google Scholar 

  26. Van den Oetelaar PJM, Mentink IM, Brinks GJ. Loss of peptides and proteins upon sterile filtration due to adsorption to membrane filters. Drug Dev Ind Pharm. 1989;1:96–106.

    Google Scholar 

  27. Matthiensen A, Beattie KA, Yunes JS, Kaya K, Codd GA. [D-Leu1] microcystin-LR, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos lagoon estuary, Brazil. Phytochemistry. 2000;55(5):383–7.

    Article  CAS  Google Scholar 

  28. Molview v2.2 online version: Accessed 16 Feb 2017.

Download references


The authors would like to thank Adam Ladak and Lauren Mullin from Waters for their support and assistance with UNIFI software.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Xavier Ortiz.

Ethics declarations

The present study did not involve human or animal participants.

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material


(PDF 133 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ortiz, X., Korenkova, E., Jobst, K.J. et al. A high throughput targeted and non-targeted method for the analysis of microcystins and anatoxin-A using on-line solid phase extraction coupled to liquid chromatography–quadrupole time-of-flight high resolution mass spectrometry. Anal Bioanal Chem 409, 4959–4969 (2017).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Microcystins
  • Anatoxin-A
  • On-line SPE
  • High throughput
  • Non-targeted
  • Data-dependent acquisition